A microfabricated fixed path length silicon sample holder improves background subtraction for cryoSAXS

The application of small-angle X-ray scattering (SAXS) for high-throughput characterization of biological macromolecules in solution is limited by radiation damage. By cryocooling samples, radiation damage and required sample volumes can be reduced by orders of magnitude. However, the challenges of reproducibly creating the identically sized vitrified samples necessary for conventional background subtraction limit the widespread adoption of this method. Fixed path length silicon sample holders for cryoSAXS have been microfabricated to address these challenges. They have low background scattering and X-ray absorption, require only 640 nl of sample, and allow reproducible sample cooling. Data collected in the sample holders from a nominal illuminated sample volume of 2.5 nl are reproducible down toq≃ 0.02 Å−1, agree with previous cryoSAXS work and are of sufficient quality for reconstructions that match measured crystal structures. These sample holders thus allow faster, more routine cryoSAXS data collection. Additional development is required to reduce sample fracturing and improve data quality at lowq.

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Limiting radiation damage for high-brilliance biological solution scattering: practical experience at the EMBL P12 beamline PETRAIII

Journal of Synchrotron Radiation ◽

10.1107/s1600577515000375 ◽

2015 ◽

Vol 22 (2) ◽

pp. 273-279 ◽

Cited By ~ 68

Author(s):

Cy M. Jeffries ◽

Melissa A. Graewert ◽

Dmitri I. Svergun ◽

Clément E. Blanchet

Keyword(s):

Radiation Damage ◽

Practical Experience ◽

Quality Data ◽

Biological Macromolecules ◽

Sample Handling ◽

X Ray ◽

X Ray Scattering ◽

Sample Position ◽

Effects Of Radiation

Radiation damage is the general curse of structural biologists who use synchrotron small-angle X-ray scattering (SAXS) to investigate biological macromolecules in solution. The EMBL-P12 biological SAXS beamline located at the PETRAIII storage ring (DESY, Hamburg, Germany) caters to an extensive user community who integrate SAXS into their diverse structural biology programs. The high brilliance of the beamline [5.1 × 1012 photons s−1, 10 keV, 500 (H) µm × 250 (V) µm beam size at the sample position], combined with automated sample handling and data acquisition protocols, enable the high-throughput structural characterization of macromolecules in solution. However, considering the often-significant resources users invest to prepare samples, it is crucial that simple and effective protocols are in place to limit the effects of radiation damage once it has been detected. Here various practical approaches are evaluated that users can implement to limit radiation damage at the P12 beamline to maximize the chances of collecting quality data from radiation sensitive samples.

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CryoSAXS as a Method for Measuring Low Resolution Macromolecular Structure

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314095916 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C408-C408

Author(s):

Jesse Hopkins ◽

Andrea Katz ◽

Stephen Meisburger ◽

Matthew Warkentin ◽

Richard Gillilan ◽

...

Keyword(s):

Radiation Damage ◽

Path Length ◽

Room Temperature ◽

Structural Information ◽

Total Sample ◽

Free Variable ◽

Low Resolution ◽

X Ray ◽

Atomic Structures ◽

Fixed Path

Small angle X-ray scattering (SAXS) is an increasingly popular technique for obtaining low resolution structural information from macromolecules and complexes in solution. Biomolecular SAXS signals can rapidly degrade due to radiation damage, so that flow or oscillating cells and large total sample volumes may be required. For particularly sensitive or hard to produce samples, such as of light sensitive proteins, metalloenzymes, and large complexes, and studies where multiple buffer conditions are probed sample consumption may be prohibitive. We describe cryo-cooling of samples to 100 K to prevent X-ray induced radiation damage. We identify SAXS-friendly cryoprotectant conditions that suppress ice formation upon cooling, and compare cryoSAXS profiles obtained in window-free variable-path-length cells with room temperature measurements for a variety of standard molecules. We obtain data sufficient for envelope reconstructions using scattering volumes as small as 20 nL, and find good agreement between cryoSAXS data and known atomic structures. We also discuss work on developing low-volume fixed path-length sample holders for cryoSAXS. Cryo-cooled samples can withstand doses that are 2-3 orders of magnitude higher than typically used for SAXS at room temperature, comparable to those used in cryo-crystallography. While practical challenges remain, cryoSAXS opens the possibility of studies exploiting high brightness X-ray sources and mail-in high-throughput SAXS. This work is funded by the NSF (DBI-1152348).

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Further application of size-exclusion chromatography combined with small-angle X-ray scattering optics for characterization of biological macromolecules

Analytical and Bioanalytical Chemistry ◽

10.1007/s00216-010-4140-7 ◽

2010 ◽

Vol 399 (4) ◽

pp. 1449-1453 ◽

Cited By ~ 9

Author(s):

Yasushi Watanabe ◽

Yoji Inoko

Keyword(s):

Size Exclusion Chromatography ◽

Small Angle ◽

Size Exclusion ◽

Biological Macromolecules ◽

X Ray ◽

X Ray Scattering ◽

Exclusion Chromatography ◽

Ray Scattering

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Upgrade of MacCHESS facility for X-ray scattering of biological macromolecules in solution

Journal of Synchrotron Radiation ◽

10.1107/s1600577514020360 ◽

2015 ◽

Vol 22 (1) ◽

pp. 180-186 ◽

Cited By ~ 40

Author(s):

Alvin Samuel Acerbo ◽

Michael J. Cook ◽

Richard Edward Gillilan

Keyword(s):

Scattering Data ◽

Biological Macromolecules ◽

Wide Angle ◽

X Ray ◽

Pristine Sample ◽

X Ray Scattering ◽

Angle Scattering ◽

Order Of Magnitude ◽

Ray Scattering

X-ray scattering of biological macromolecules in solution is an increasingly popular tool for structural biology and benefits greatly from modern high-brightness synchrotron sources. The upgraded MacCHESS BioSAXS station is now located at the 49-pole wiggler beamline G1. The 20-fold improved flux over the previous beamline F2 provides higher sample throughput and autonomous X-ray scattering data collection using a unique SAXS/WAXS dual detectors configuration. This setup achieves a combinedq-range from 0.007 to 0.7 Å−1, enabling better characterization of smaller molecules, while opening opportunities for emerging wide-angle scattering methods. In addition, a facility upgrade of the positron storage ring to continuous top-up mode has improved beam stability and eliminated beam drift over the course of typical BioSAXS experiments. Single exposure times have been reduced to 2 s for 3.560 mg ml−1lysozyme with an average quality factorI/σ of 20 in the Guinier region. A novel disposable plastic sample cell design that incorporates lower background X-ray window material provides users with a more pristine sample environment than previously available. Systematic comparisons of common X-ray window materials bonded to the cell have also been extended to the wide-angle regime, offering new insight into best choices for variousq-space ranges. In addition, a quantitative assessment of signal-to-noise levels has been performed on the station to allow users to estimate necessary exposure times for obtaining usable signals in the Guinier regime. Users also have access to a new BioSAXS sample preparation laboratory which houses essential wet-chemistry equipment and biophysical instrumentation. User experiments at the upgraded BioSAXS station have been on-going since commissioning of the beamline in Summer 2013. A planned upgrade of the G1 insertion device to an undulator for the Winter 2014 cycle is expected to further improve flux by an order of magnitude.

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Quantifying radiation damage in biomolecular small-angle X-ray scattering

Journal of Applied Crystallography ◽

10.1107/s1600576716005136 ◽

2016 ◽

Vol 49 (3) ◽

pp. 880-890 ◽

Cited By ~ 24

Author(s):

Jesse B. Hopkins ◽

Robert E. Thorne

Keyword(s):

Radiation Damage ◽

Small Angle ◽

Structural Information ◽

Systematic Investigation ◽

Radius Of Gyration ◽

Biological Macromolecules ◽

X Rays ◽

X Ray ◽

X Ray Scattering ◽

Ray Scattering

Small-angle X-ray scattering (SAXS) is an increasingly popular technique that provides low-resolution structural information about biological macromolecules in solution. Many of the practical limitations of the technique, such as minimum required sample volume, and of experimental design, such as sample flow cells, are necessary because the biological samples are sensitive to damage from the X-rays. Radiation damage typically manifests as aggregation of the sample, which makes the collected data unreliable. However, there has been little systematic investigation of the most effective methods to reduce damage rates, and results from previous damage studies are not easily compared with results from other beamlines. Here a methodology is provided for quantifying radiation damage in SAXS to provide consistent results between different experiments, experimenters and beamlines. These methods are demonstrated on radiation damage data collected from lysozyme, glucose isomerase and xylanase, and it is found that no single metric is sufficient to describe radiation damage in SAXS for all samples. The radius of gyration, molecular weight and integrated SAXS profile intensity constitute a minimal set of parameters that capture all types of observed behavior. Radiation sensitivities derived from these parameters show a large protein dependence, varying by up to six orders of magnitude between the different proteins tested. This work should enable consistent reporting of radiation damage effects, allowing more systematic studies of the most effective minimization strategies.

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Automated Pipeline for Purification, Biophysical and X-Ray Analysis of Biomacromolecular Solutions

Scientific Reports ◽

10.1038/srep10734 ◽

2015 ◽

Vol 5 (1) ◽

Cited By ~ 65

Author(s):

Melissa A. Graewert ◽

Daniel Franke ◽

Cy M. Jeffries ◽

Clement E. Blanchet ◽

Darja Ruskule ◽

...

Keyword(s):

Biological Macromolecules ◽

Analysis Pipeline ◽

X Ray ◽

X Ray Scattering ◽

Automated Pipeline ◽

Automated Data Analysis ◽

Sample Component ◽

Data Analysis Pipeline ◽

Line Sample

Abstract Small angle X-ray scattering (SAXS), an increasingly popular method for structural analysis of biological macromolecules in solution, is often hampered by inherent sample polydispersity. We developed an all-in-one system combining in-line sample component separation with parallel biophysical and SAXS characterization of the separated components. The system coupled to an automated data analysis pipeline provides a novel tool to study difficult samples at the P12 synchrotron beamline (PETRA-3, EMBL/DESY, Hamburg).

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Characterization of defects in tabular silver halide grains

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100178367 ◽

1990 ◽

Vol 48 (4) ◽

pp. 1046-1047

Author(s):

C. Goessens ◽

D. Schryvers ◽

J. Van Landuyt ◽

A. Verbeeck ◽

R. De Keyzer

Keyword(s):

Radiation Damage ◽

Silver Halide ◽

Chemical Characteristics ◽

X Ray ◽

Free Region ◽

Central Defect ◽

Crystallographic Defects ◽

Physical And Chemical ◽

Two Sides

Silver halide grains (AgX, X=Cl,Br,I) are commonly recognized as important entities in photographic applications. Depending on the preparation specifications one can grow cubic, octahedral, tabular a.o. morphologies, each with its own physical and chemical characteristics. In the present study crystallographic defects introduced by the mixing of 5-20% iodide in a growing AgBr tabular grain are investigated. X-ray diffractometry reveals the existence of a homogeneous Ag(Br1-xIx) region, expected to be formed around the AgBr kernel. In fig. 1 a two-beam BF image, taken at T≈100 K to diminish radiation damage, of a triangular tabular grain is presented, clearly showing defect contrast fringes along four of the six directions; the remaining two sides show similar contrast under relevant diffraction conditions. The width of the central defect free region corresponds with the pure AgBr kernel grown before the mixing with I. The thickness of a given grain lies between 0.15 and 0.3 μm: as indicated in fig. 2 triangular (resp. hexagonal) grains exhibit an uneven (resp. even) number of twin interfaces (i.e., between + and - twin variants) parallel with the (111) surfaces. The thickness of the grains and the existence of the twin variants was confirmed from CTEM images of perpendicular cuts.

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Small angle X-ray scattering study of porosity variation in a styrene-divinylbenzene copolymer

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19811675 ◽

1981 ◽

Vol 46 (7) ◽

pp. 1675-1681 ◽

Cited By ~ 13

Author(s):

Josef Baldrian ◽

Božena N. Kolarz ◽

Henrik Galina

Keyword(s):

Small Angle ◽

Structural Parameters ◽

Two Phase ◽

X Ray ◽

Porosity Variation ◽

Swelling Agent ◽

X Ray Scattering ◽

Styrene Divinylbenzene ◽

Ray Scattering

Porosity variations induced by swelling agent exchange were studied in a styrene-divinylbenzene copolymer. Standard methods were used in the characterization of copolymer porosity in the dry state and the results were compared with related structural parameters derived from small angle X-ray scattering (SAXS) measurements as developed for the characterization of two-phase systems. The SAXS method was also used for porosity determination in swollen samples. The differences in the porosity of dry samples were found to be an effect of the drying process, while in the swollen state the sample swells and deswells isotropically.

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